The present invention relates to methods of identifying and isolating genes which are involved in the regulation of fungal gene expression. The invention also relates to methods useful for identifying fungal virulence factors. It further relates to a method of identifying agents which increase or decrease the expression or activity of a gene that regulates or is required for fungal pathogenesis. The invention also relates to the use of such agents as fungicides or fungistats.
Fungi are a large and diverse group of organisms with enormous importance to humans. Pathogenic fungi are a significant cause of human disease, particularly in the rapidly increasing proportion of the population whose immune system has been compromised by disease, chemotherapy, or immunosuppressive drugs. A wide variety of plant-pathogenic fungi (e.g., blights, rusts, molds, smuts, mildews) cause huge food crop loss and damage to ornamental plants. Plant diseases are caused by a myriad of invasive fungal pathogens falling into many genera, for example: soft rot (e.g., Rhizopus), leaf curl (e.g., Taphrina), powdery mildew (e.g., Sphaerotheca), leaf spots (e.g., Fulvia), blight (e.g., Alternaria), blast (e.g., Magnaporthe), black rot (e.g., Guignardia), scab (e.g., Venturia), wilts (e.g., Fusarium), rusts (e.g., Puccinia), smuts (e.g., Ustilago), and cankers (e.g., Rhizoctonia). In addition, fungal species are the commercial source of a great many medicinally useful products, such as antibiotics (e.g., beta-lactam antibiotics such as penicillin, cephalosporin, and their derivatives), anti-hypercholesterolemic agents (e.g., lovastatin and compactin), immunosuppressives (e.g., cyclosporin), and antifungal drugs (e.g., pneumocandin and echinocandin). All of these drugs are fungal secondary metabolites, small secreted molecules that fungi utilize against competitors in their microbial environment. Finally, fungi also produce commercially important enzymes (e.g., cellulases, proteases, and lipases) as well as other products (e.g., citric acid, gibberellic acid, natural pigments, and flavorings).
The specifics by which fungi invade their growth substrate are not understood in detail. However, two important themes regarding the fungal invasion process have emerged in recent years. First, important human fungal pathogens, such as Candida sp., Aspergillus sp., Mucor sp., Rhizopus sp., Fusarium sp, Penicillium marneffei, Microsporum sp. and Trichophyton sp. invade through host tissues as filamentous hyphae. The virulence of Candida (C.) albicans has been shown to be dependent upon invasion of host tissues; mutations in any of several genes required for invasive growth substantially reduce virulence in a mouse model of systemic infection. Pathogenesis of the plant fungal pathogen Ustilago (U.) maydis also requires invasion. Second, there is a correlation between genes that regulate agar invasion in Saccharomyces (S.) cerevisiae and genes that control invasion in pathogenic yeast. As S. cerevisiae is amenable to genetic studies, it can be utilized to molecularly dissect the genetics of fungal invasion.
Homologs of certain S. cerevisiae genes required for invasion also regulate the production of secondary metabolites and secreted catabolic enzymes in other fungi. For example, activating mutations in Aspergillus homologs of the S. cerevisiae INV genes cause increased production of the secondary metabolite penicillin and a secreted alkaline phosphatase (Orejas et al., Genes Dev. 1995, 9:1622).